Acute Toxin Standard Operating Procedures
For working with:
Toxin Name:
Building/Room:
P.I. Name:
EH&S Approval:
Revision #:
Effective Date:
P.I. Signature:
Approval Date:
Note: The UF EH&S Biosafety Office oversees the proper acquisition, use, storage and disposal of unfractionated
mixtures and purified preparations of biological toxins with a mammalian LD50 ≤ 100µg/kg body weight (referred to
as Acute Toxins), as well as natural and recombinant organisms which produce these biological toxins. All research
projects involving an acute toxin(s) must be registered with the Biosafety Office using the Acute Toxin Registration
Form.
This document is intended to be adapted to the lab as a site-specific document. Additional precautions or
regulations may be required per risk assessment by the Biological Safety Office and/or Institutional Biosafety
Committee and/or Responsible Official.
This SOP must be submitted to the Biosafety Office with your Acute Toxin Registration Form and must be approved
by the UF EH&S Biosafety Office BEFORE work can begin with the toxin. If you are working with multiple toxins,
you must indicate site-specific and toxin-specific procedures if you choose to use a single SOP to cover all toxins
used in your lab.
This SOP should be revised whenever significant changes to the experimental protocol are made and/or at least
annually. All revisions and annual reviews will require approval by the EH&S Biosafety Office and must be read,
understood and signed off by all personnel working with the toxin.
1. Circumstances of Use:
See “Instructions for Creating a Lab-Specific SOP.”
2. Potential Hazards:
Routes of Exposure:
Target Organs:
Signs/Symptoms of Exposure:
Incubation period:
Toxicological data (by route of entry):
Fill in the above information. See “Instructions for Creating a Lab-Specific SOP” for additional guidance.
3. Medical Considerations:
See “Instructions for Creating a Lab-Specific SOP.”
4. Security Procedures:
The following security measures are required for work with Acute Toxins:
 Only minimum amounts of toxin(s) will be kept on hand.
 All toxin orders should be sent in such a manner in which the package can be tracked and requires your
signature for receipt (we strongly recommend using FedEx).
 Toxin stocks and solutions must be secured at all times when not in use.
o Toxin is stored in: (fill in equipment and storage site)
o The key or combination to the lock is under the control of the P.I.
o If a lock box is used, the box must be affixed to the refrigerator or freezer or the refrigerator or
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freezer must also be locked.
Toxin Inventory:
o A hardbound log book must be used to record the inventory. Each batch of toxin should be recorded
on a separate page (see Appendix A for sample log book entry).
 The following information should be recorded at the top of the page: Toxin Name, Date
Received, Source or Received From, Amount Received, Form Received (Liquid or Powder),
Concentration of Liquid Stock, Total Volume
 The following headings should be used to track the Removal/Use of Toxin: Starting Amount,
Amount Removed/Used, Date of Removal/Use, Purpose, Removed by (Print Name),
Signature and Amount Remaining.
o The inventory book should also contain a section to record the destruction/inactivation of toxin stock.
 The following headings should be used: Toxin Name, Date Destroyed, Amount Destroyed,
Method of Destruction, Destroyed by (Print Name), Signature, Witnessed by (Print Name),
Signature, Amount remaining (if any)
o All entries in the book shall be done in ink.
o The Inventory book must be kept in a secure location (e.g., locked drawer) and the key or
combination shall remain under the control of the P.I.
o Discrepancies, alterations, or suspicious entries must be reported to the P.I. and the Biological Safety
Office (392-1591) immediately.
o EH&S staff will inspect the inventory and log books on a periodic basis.
Add additional security practices following the guidance provided in “Instructions for Creating a Lab-Specific SOP.”
5. Administrative Controls:
 All non-essential personnel must be excluded from the work area when toxins are in use.
o Post the “Toxins in Use” sign (see Appendix B) to warn/exclude personnel not directly involved in the
toxin work.
 All personnel working with the toxins should be familiar with the MSDS sheet for each toxin and signs and
symptoms of exposure.
 The “buddy system” is required in the following instances so that assistance is immediately available if
needed:
o Work with concentrations or amounts near or exceeding the LD50 dose for a human
o High risk activities with acute toxins including:
 Procedures that generate or potentially generate aerosols (e.g., vortexing, grinding,
centrifuging, intra-nasal inoculation of animals, sonication, etc.)
 Utilization of concentrated stocks or large quantities of toxins
 Work with powdered or dried toxins
 Any work with highly lethal toxins or highly purified forms of less lethal toxins
 Use of needles or sharps in experiments with toxin
o Based on any recommendation provided by the Biosafety Office, IBC and/or RO/ARO.
6. Engineering Controls:
 All toxins should be handled in the operationally effective zone of a certified biological safety cabinet (BSC) or
fume hood for all procedures where aerosols may be produced
o Aerosols may be produced during routine laboratory procedures (e.g., vortexing, mixing, pipetting)
and during open or pressurized manipulations of suspensions.
Add additional engineering controls using the guidance provided in “Instructions for Creating a Lab-Specific SOP.”
7. Safe Work Practices:
 All packages containing the toxin should be opened in a certified biosafety cabinet or fume hood.
o Inspect the toxin container for damage during shipping.
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If damaged, decontaminate and dispose of immediately following procedures established in
Section 10: Waste Disposal; contact vendor.
 If not damaged, packaging may be discarded in the regular trash.
o Log receipt of toxin shipment immediately in the Toxin Log book and secure the toxin.
Experiments should be designed to eliminate or minimize work with dry toxins.
o Liquid stocks should be purchased when available since toxin in powdered form is more hazardous
than toxin in liquid form.
Minimize the use of sharps; safety sharps should be considered (and may be required per risk assessment). At
a minimum:
o Luer-lock syringes are required
o Glass Pasteur pipettes are prohibited.
Set up the work area with the appropriate equipment prior to the start of all experiments:
o Place a blue absorbent, plastic-backed pad sprayed with an appropriate disinfectant in the work area
of the BSC (be careful not to block the front or rear grills).
 Conduct all work with toxin over the disinfectant-soaked pad..
o Ensure that containers for solid waste (pipette tips, etc) and/or liquid waste (filled with appropriate
disinfectant) are accessible inside the BSC or fume hood.
o Toxin-specific spill kit is readily accessible
o Sharps containers are accessible within BSC or fume hood (if required)
o All PPE
Disposable materials should be used whenever possible for work with toxins; containers that will be re-used
will require extensive decontamination prior to washing.
The interior of the BSC or fume hood and all equipment (e.g., pipettors) should be thoroughly decontaminated
after work with toxins with (list appropriate disinfectant and concentration).
Remove and dispose of, or decontaminate, PPE and wash hands with soap and water before leaving the work
area.
Add additional precautions following the guidance provided in “Instructions for Creating a Lab-Specific SOP.”
8. Personal Protective Equipment (PPE):
 Long pants and full-coverage shoes are required for work with toxins.
 A laboratory coat or gown must be worn at all times when working with toxins. Cloth lab coats may not be
taken home for laundering; they must first be autoclaved prior to transport to a UF-approved laundering
service. Disposable lab coats are strongly recommended.
 Safety glasses with side shields or goggles should be worn when working with toxins.
o Safety glass should be decontaminated after each use with an appropriate disinfectant prior to reuse.
 Two pairs of disposable latex and/or nitrile gloves should be worn when working with toxins.
o Alternating nitrile gloves as the inner pair and latex gloves the outer pair will make glove changes
easier.
o Contaminated gloves should be removed carefully as soon as possible and placed into an appropriate
disinfectant or autoclavable bag that is then closed. A new pair of outer gloves is donned.
 Personnel responding to spills outside of a biocontainment device are required to wear all of the above PPE
and an N95 respirator. The use of a respirator requires both a medical clearance and a fit test to be worn.
Contact UF EH&S at 392-1591 for more information.
Add additional PPE requirements following the guidance provided in “Instructions for Creating a Lab-Specific SOP.”
9. Storage, Transportation and Shipping:
 When stored in the laboratory, toxin containers must be sealed, legibly labeled (toxin name and hazard
warning at a minimum) and secured (see Section 4: Security Procedures) to ensure restricted access.
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Individual tubes of toxin dilutions should also be stored in sealed, labeled secondary containers (e.g.,
Eppendorf tubes of toxin dilutions can be placed in a 50ml conical tube that is appropriately labeled
with toxin name, concentration and biohazard sticker).
Refrigerators and/or other storage containers housing biological toxins should be labeled with contact
information for trained, responsible laboratory personnel.
Toxins and toxin solutions should be transported in closed, double or triple containment (e.g., small tubes
within a large conical tube inside a Tupperware container, or a vial in a sealed bag) with absorbent and
cushioning material.
All storage and transport containers must be labeled with a biohazard sticker.
Transport of toxins should occur through low-traffic hallways whenever possible.
Shipping biological toxins requires current training certification in IATA/ICAO and DOT regulations. Contact
the UF Biosafety Office (392-1591) for more information.
Add additional precautions following the guidance provided in “Instructions for Creating a Lab-Specific SOP.”
10. Decontamination and Waste Disposal:
Decontamination – the following information must be included (at least one method listed):
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Name and concentration of decontamination solution: fill in
Time for effective decontamination: fill in
Physical inactivation (method, temperature, time): fill in
Disposal of Toxin-Contaminated, or Potentially Contaminated Liquid Waste
 For collection of liquid waste during a toxin experiment, set up a liquid waste container in the BSC.
o The container should be a durable, disposable, leak-proof container.
 For example, for small amounts of liquid waste- a 15ml or 50ml conical tube could be
used to collect liquid waste.
o Add an appropriate amount of (insert decontamination solution, 2X concentration) to the
container and decant or pipet liquid waste into the container during the experiment.
 Care should be taken to minimize aerosols and splashes. Dispense liquid waste from a
pipet by holding the pipet tip against the side of the collection container or submerging
the tip directly in the disinfectant solution.
 At the completion of the experiment, place the primary waste container into a secondary, durable, leakproof container and seal.
o Fill out a yellow hazardous (chemical) waste label indicating concentration of components in the
waste bottle (including the amount and concentration of bleach) and affix to the bottle.
o Store the doubly-contained inactivated toxin in the designated hazardous waste accumulation
area (fill in location). Do not store toxin waste for prolonged periods of time. Toxin waste
should be picked up ASAP as a best management practice.
 To schedule a hazardous waste pick up call EH&S (392-8400) or schedule on-line. EH&S
Hazardous Waste will not pickup toxin materials that have not been inactivated.
Disposal of Toxin-Contaminated, or Potentially Contaminated Solid Waste
See “Instructions for Creating a Lab-Specific SOP.”
Decontamination/Cleaning of Toxin-Contaminated, or Potentially Contaminated Reusable Labware
See “Instructions for Creating a Lab-Specific SOP.”
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11. Inactivation and Disposal of Liquid Toxin Stock Solutions:
Note: All destruction or inactivation events of concentrated toxin stock solutions require a witness and must be
documented in the Inventory Log. Destruction/Inactivation of select agent toxin stock material associated with a
select agent-registered project must be witnessed and documented by the UF RO/ARO. For all other
destruction/inactivation events, the witness may be a second member of the laboratory approved for toxin use.
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Donn PPE as described in section 8: Personal Protective Equipment.
Place plastic-backed absorbent pad in the working area of the BSC (be careful not to block the front or
rear grill) and spray with disinfectant.
Place primary toxin container in a secondary plastic disposable container with a sealable lid.
Carefully add an equal volume of (insert decon solution, concentration) into the toxin solution.
Allow a minimum of 1 hr contact time to inactivate the toxin.
o Replace cap on primary container and secure the lid on secondary container.
o Fill out a yellow hazardous (chemical) waste label indicating concentration of components in the
waste bottle (including the amount and concentration of bleach) and affix to the bottle.
o Store the doubly-contained inactivated toxin in the designated hazardous waste accumulation
area (fill in location). Do not store toxin waste for prolonged periods of time. Toxin waste
should be picked up ASAP as a best management practice.
o To schedule a hazardous waste pick up call EH&S (392-8400) or schedule on-line. EH&S
Hazardous Waste will not pickup toxin materials that have not been inactivated.
12. Spill Response:
Spill Response:
1. Spills of acute toxins inside BSC (any volume, concentration):
 Note: If the spill extends into the airflow grill and drain pan of the BSC, contact the PI, BSO or EH&S before
proceeding.
 Remove any contaminated PPE/clothing and dispose of in a biohazard bag; close bag.
o If you are contaminated- immediately wash the affected area with soap and water.
o Replace PPE and alert others in area to spill.
 Cover spill with paper towels or other absorbent material.
 Apply (list decon solution and concentration) to the spill, starting at the perimeter and moving towards
the center of the spill area.
 Allow disinfectant to sit for a minimum of 30 minutes.
 Pick up absorbent materials and wipe up excess decontamination solution; dispose of in a biohazard bag.
Use tweezers, forceps or tongs to pick up sharps and place in sharps container.
 Wipe walls and/or cabinets that may have been splashed with (List decon solution and concentration).
 Rinse all decontaminated surfaces with water followed by 70% Ethanol. Dispose of paper towels in
appropriate waste container.
 Follow the appropriate Waste Disposal Procedures as outlined in Section 10: Decontamination and Waste
Disposal.
 Wash hands and exposed skin with soap and water before exiting laboratory.
 Notify PI of the spill. If appropriate, the PI will inform the Biological Safety Officer.
 Report any potential exposure to the Student Health Care Center.
2. Spills of acute toxins outside the BSC (any volume, concentration):
 Alert all personnel in the room of the spill and immediately evacuate to the hallway.
o Remove any contaminated PPE at the doorway to the hall, if possible.
o Decontaminate shoes, if necessary.
o If you are contaminated, immediately wash the affected area with soap and water.
 Secure the area and post a “Spill: Do Not Enter” sign on the laboratory door.
 Immediately notify the PI and the Biological Safety Officer or EH&S of the spill
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o Do not re-enter the laboratory until you have consulted with biosafety personnel.
o Prior to re-entry into the contaminated lab, Biosafety personnel will develop and implement a
decontamination and clean-up plan.
Report any potential exposure to the Student Health Care Center.
13. Exposure Management:
Location of nearest Emergency Eyewash: fill in location
Location of nearest Emergency Shower: fill in location
Skin or Wound Exposures:
 Move to (or move person to) an uncontaminated area.
 Remove contaminated clothing.
 Wash skin and wound exposures with fresh 0.5% sodium hypochlorite or soap and water.
 For spills of large quantities of toxin onto clothing or skin, immediately use the emergency shower and follow
with fresh 0.5% sodium hypochlorite or soap and water.
o Place contaminated clothing in a red biohazard bag for decontamination.
Eye or Mucous Membrane Expsosure:
 Use emergency eyewash to immediately flush eyes with gently flowing, potable water for a minimum of 15
minutes
o Forcibly hold eye open to ensure effective rinsing behind eyelids. Move eyes side-to-side and up-anddown during rinsing
o Remove contact lenses.
In all instances:
 Obtain medical attention as required:
o Working hours: Student Health Care Center (SHCC) on main campus or satellite SHCC in Dental Tower
(D2-52)
o After-hours: Call 911 or go to the nearest medical facility
 Review the MSDS for symptoms of exposure or delayed onset effects.
 Report Incident to Supervisor (insert supervisor and phone #) and Biological Safety Office (392-1591).
 Contact UF Worker’s Compensation (352-392-4940).
 Follow spill cleanup procedures (Section 13) for contaminated surfaces and materials.
14. Personnel Training:
In addition to General Biosafety Training, all laboratory personnel working with acute toxins must be trained in the
theory and practice of the toxin being used with special emphasis on the nature of the practical hazards associated
with laboratory operations; this includes how to handle transfers of liquids containing the toxin, waste disposal,
decontamination of materials, equipment and work surfaces after routine laboratory operations and appropriate
spill response procedures. Furthermore, all personnel must read and fully adhere to this SOP when handling the
toxin.
Add any additional laboratory-specific training required for work with toxins.
15. Animal Use of Biotoxins
Please check one of the following:
☐ We will not be dosing animals with the toxin.
☐ We will be dosing animals with the toxin.
If you checked the second box and will be dosing animals with the toxin, the “Animal Use of Biotoxins” section
(Appendix C) must be completed and followed. The Animal section includes requirements related to notification
and coordination of Animal Care Services (ACS), room and cage labeling and health considerations for animal
caretakers.
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“I have read and understand this SOP. I agree to fully adhere to its requirements.”
Last Name
First Name
UFID#
Signature
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Instructions for Creating a Laboratory-Specific Standard Operating Procedure (SOP) for Acute Toxins
Use fillable form starting on page 1 to create your lab-specific SOP. Include relevant information from all sections.
Risks should be assessed for each toxin individually to determine the appropriate controls, personal protective
equipment, and decontamination procedures for preparing, handling and disposing of the toxin.
These instructions are meant to provide guidance in creating your laboratory-specific SOP and may not address
every possible safety and/or security concern. You should use these instructions, along with your toxin risk
assessment and any guidance from the Biosafety Office to ensure that you address all potential hazards and means
of mitigating those hazards.
1. Circumstances of Use:
Provide a brief description of the work and what the toxin will be used for. Include the following in your
description:
 Quantity required, approximate frequency of use and location(s) of use.
o For select agent toxins- ensure that the cumulative amount of toxin under the control of a PI
does not exceed the permissible toxin amounts.
 Describe concentrations used and how needed concentration and amount will be prepared.
 Indicate whether the toxin will be purchased in liquid or powder form.
o Liquids are generally less hazardous than powders; purchase acute toxins in liquid form if
possible.
o Consider purchasing dilute solutions when possible.
2. Potential Hazards:
Fill in the required information for each toxin.
 Possible routes of entry:
o Consider: skin, inhalation, mucous membranes, ingestion, auto-inoculation, etc.
o For each potential route of exposure- indicate how/when exposure might occur (e.g., inhalationduring weighing, mixing or splashes; needle sticks from injection preparation and administration,
etc).
 The following toxins have summary statements in the Toxin Agent section of the 5th edition of the
NIH/CDC’s Biosafety in Microbiological and Biomedical Laboratories manual which may be helpful:
o Botulinum neurotoxin
o Staphylococcal enterotoxins
o Ricin toxin
o Selected low molecular weight toxins
 T-2 mycotoxin
 Saxitoxin and related paralytic shellfish poisons
 Tetrodotoxin
 Brevetoxin
 Palytoxin
 Polypeptide conotoxins
 Microcystin-LR
 The Toxin, Toxin-Target Database has detailed toxin information that may also be helpful in determining
potential hazards.
 If you are having difficulty finding literature or guidance for your particular toxin, contact the EH&S
Biosafety Office for assistance (392-1591).
3. Medical Considerations:
 List any toxoid vaccinations or antitoxins required or recommended for this toxin. Consult with EH&S
Biosafety Office if you need assistance.
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If antitoxins or toxoid vaccinations are required, all personnel planning to work with the toxin
must contact the Student Health Care Center at 352-294-5700 to discuss these considerations.
If there are no specific medical considerations recommended for work with the toxin, please put “No
special medical considerations recommended.”
4. Security Procedures:
If you are working with any amount of a select agent toxin, you must adhere to the Toxin Due Diligence Provision
for Select Agent Toxins. 42 CFR 73.3(d) requires that select agent toxins may only be transferred after the
transferor uses due diligence and documents that the recipient has a legitimate need (i.e., reasonably justified by a
prophylactic, protective, bona fide research, or other peaceful purpose) to handle or use the toxin.
If you are working with a select agent toxin- copy and paste the following into the Section 4: Security –
 All PIs in possession of any quantity of a Select Agent Toxin must retain documentation of all
transfers of any quantity of the toxin outside of the laboratory; documentation should be retained by
the PI for a minimum of three (3) years. The required documentation must include the following:
o Name of the recipient, institution, address, telephone # and email address;
o Toxin and quantity transferred;
o Purpose of use/knowledge of recipient’s legitimate need for the toxin.
 This can be obtained either by requiring the recipient to complete documentation
stating their intended use of the toxin OR by the transferor documenting their
knowledge of the recipients legitimate need for the toxin.
 Reporting Suspected Violations or Suspicious Activity. If a PI in possession of any quantity of a
Select Agent Toxin detects suspicious activity associated with a request for toxin or suspicious activity
associated with a shipped toxin, s/he must immediately notify UF’s Responsible Official (RO) or
Alternative Responsible Official (ARO) who will then contact the Federal Select Agent Program.
o Alternatively, anonymous reporting is possible through the Office of the Inspector General:
 Anonymous OIG Hotline: 1-800-HHS-TIPS (800-447-8477)
 Web: http://oig.hhs.gov/fraud/hotline
 When reporting issues to the OIG, ensure that you indicate this is a Select Agent
Program Issue.
5. Administrative Controls:
Add any additional administrative controls per your toxin risk assessment or the recommendation of the Biosafety
Office.
6. Engineering Controls:
Engineering controls should be selected according to the risk assessment for each specific toxin operation. List the
engineering controls required for this toxin (e.g., HEPA filtered enclosure inside chemical fume hood or Class III
Biological Safety Cabinet may be required for experiments that intentionally aerosolize toxin).
Resources that may be useful in determining appropriate engineering controls include:
 Appendix I of the BMBL, 5th edition: Guidelines for Work with Toxins of Biological Origin
 Section VIII-G: Toxin Agents of the BMBL, 5th edition
 “Safety and Health Considerations for Conducting Work with Biological Toxins.” Applied Biosafety, 6(3)
pp. 117-135, 2001.
 EH&S Biosafety Office (392-1591)
Things to consider when listing Engineering controls for toxins (include all that apply):
 Intentional generation of aerosols is considered a high-risk operation that requires approval by the
Biosafety Office (392-1591) to ensure that appropriate engineering controls are in place.
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Low molecular weight (LMW) toxins or work involving radionucleotides combined with toxins may require
the use of a charcoal-filter in addition to HEPA filtration.
Work with toxins involving volatile chemicals requires a fully exhausted BSC or use of secondary
containment (glove bag or box) inside a fume hood.
Use of powdered toxins should be avoided if possible and minimized otherwise.
o All manipulations with powdered toxins in open vials must be conducted in a Class III BSC (no
secondary containment required) or in a Class II BSC or fume hood with the use of secondary
containment (e.g., a disposable glove bag or box inside).
o Respirator use may be required even for work inside a BSC for toxins with a low LD 50 or LC50 for
protection in the event of engineering control failure.
Operations that expose toxin solutions to vacuum or pressure should always be conducted within a
certified fume hood or BSC
o Respiratory protection may be required to protect personnel in case of engineering failure.
Contact the Biological Safety Officer (BSO) for guidance on respiratory protection requirements.
o If vacuum lines are used during toxin manipulations, they must be protected by an in-line HEPA
filter to prevent contamination of the building vacuum system with toxin.
7. Safe Work Practices:
If animals will be dosed with the toxin, the “Animal Use of Biotoxins” section (Appendix C) must be completed
and followed. The Animal section includes requirements related to notification and coordination the Animal
Care Services (ACS), room and cage labeling and health considerations for animal caretakers.
Things to consider when listing Safe Work Practices for toxins (include all that apply):
 If powdered stocks are purchased, rehydration of the powder stock must occur in a certified BSC due to
possible pressure changes that may generate aerosols through the vent opening.
o Rehydrate the powder through the septum without opening the vial.
o Care should be taken not to over-pressurize the vial.
o Cover/shield the vial stopper with a kimwipe soaked in (list decontamination solution).
o Use extreme care when handling sharps.
 Pressurized tubes containing toxins should only be opened in a certified BSC with a HEPA filter.
 Safety sharps devices and safety needles should be used to minimize risk of cuts or punctures. Refer to
the Safety Engineered Sharps handout on the UF Biosafety webpage for examples and ordering
information.
 Centrifugation of toxin solutions requires the use of sealed rotors or centrifuge safety cups.
o Rotors and safety cups should be loaded and unloaded in the BSC.
o Exterior surfaces of the primary containers should be wiped with decontamination solution prior
to being placed in rotor or safety cups.
o Exterior surfaces of rotors or safety cups should be wiped with decontamination solution prior to
being removed from the BSC.
o After use, centrifuge rotors or safety cups should be decontaminated and rinsed with water or
70% Ethanol.
8. Personal Protective Equipment (PPE):
Describe additional PPE requirements for each task involving the toxin.
Things to consider when listing PPE for toxins (include all that apply):
 If a toxin is suspended in a solvent, appropriate gloves should be worn to prevent penetration of the
solvent. Solvents can act as carriers for the toxin resulting in dermal exposure if the solvent readily
penetrates the glove.
o Good references for chemical-glove compatibility include:
 Chemical Guide and Permeation Tables found on the EH&S website of Oklahoma State
University
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 Showa Best Gloves Chemrest Chemical Guide
If there is a potential risk of cut or puncture, cut- or puncture-resistant gloves should be worn. See the
Safety-Engineered Sharps handout on the UF Biosafety Webpage for examples and ordering information.
When conducting liquid transfers or other operations that pose a potential splash or droplet hazard in a
Class II BSC or fume hood, safety goggles and disposable facemask or face shield should be worn.
If working with dry forms of toxin that are subject to spread by electrostatic dispersal, static-free
disposable gloves must be worn. Wetting and wiping down gloves and surfaces before beginning work
can also help reduce static.
Note on Respirators: Respirators are masks designed to protect the wearer from specific airborne hazards
and are different from surgical masks, which protect the wearer only from splashes and are primarily
intended to protect others from infectious aerosols exhaled by the wearer. Respirator use requires
employee participation in the Respiratory Protection Program, which involves medical clearance and
annual fit testing.
o It is strongly recommended that all personnel working with toxins participate in the Respiratory
Protection Program and be fit-tested for an N95 respirator; at a minimum, this will ensure that
personnel are prepared to handle a spill cleanup if necessary.
9. Storage, Transportation and Shipping:
Describe any additional controls per your toxin risk assessment and/or the recommendation of the Biosafety
Office.
10. Decontamination and Waste Disposal:
Describe the waste disposal procedures taking the following into consideration:
 Discarded needles/syringes and other sharps should be placed directly into properly labeled, punctureresistant sharps containers, and inactivated/decontaminated as soon as practical.
 Depending on the toxin, contaminated materials and toxin waste solutions may be inactivated by physical
means (incineration or extensive autoclaving), or by chemical means (soaking in suitable decontamination
solutions for a specified period of time).
o To determine the appropriate physical and/or chemical means of inactivating your particular
toxin, consult one or more of the following:
 University of Virginia Biotoxin Inactivation Table
 Tables 1 and 2 of the NIH/CDC’s BMBL Guidelines for Work with Toxins of Biological
Origin
 The MSDS of your toxin
Use the following sample protocols as a template for the waste disposal procedures for your toxin:
 Sample protocol for disposal of solid waste and re-useable labware contaminated with toxins that can
be inactivated by autoclaving:
o Contaminated, or potentially contaminated items (gloves, pipet tips, eppendorf tubes, paper
towels, etc), are collected in a red biohazard-labeled autoclavable bag in the BSC in a durable,
leak-proof container.
 At completion of the experiment, the bag is closed and sprayed with freshly prepared
(insert decontamination solution, concentration) and allowed to sit for a minimum of 5
minutes before removing from the BSC and transport to the autoclave.
 Contaminated PPE should also be disposed of in a red biohazard-labeled autoclavable
bag and autoclaved.
o Autoclave the material using a biohazard cycle on the autoclave.
 Recommended cycle for heat-labile toxins: 121°C, 15psi for a minimum of 1 hr.
o At the completion of the cycle, open the autoclave slowly taking care not to breathe in the
steam; allow autoclave to vent before collecting the waste from the autoclave.
o Place the autoclaved bag into the biological waste box for disposal as biowaste.
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
Sample protocol for disposal of solid waste contaminated with toxins that cannot be inactivated by
autoclaving:
o Contaminated, or potentially contaminated items (gloves, pipet tips, eppendorf tubes, paper
towels, etc), are collected in a red biohazard-labeled bag in the BSC in a durable, leak-proof
container.
 At completion of the experiment, the bag is closed and sprayed with freshly prepared
(insert decontamination solution, concentration) and allowed to sit for a minimum of 5
minutes before removing from the BSC.
 Additional contaminated, or potentially contaminated PPE (e.g., disposable gowns) is
also collected in a red, biohazard-labeled bag and closed at the completion of the
experiment.
 Place closed bags into a biowaste box lined with a red, biohazard-labeled bag. These
boxes are picked up by Stericycle for incineration.

Sample protocol for decontamination/cleaning of re-useable labware contaminated with toxins that
cannot be inactivated by autoclaving:
o The use of reusable labware for work with toxins should be eliminated whenever possible and
minimized if not possible.
o Re-usable labware (e.g., glass tubes, glass beakers/flasks, etc) will be submerged in freshly
prepared (insert decontamination solution, concentration) for a minimum of (insert time) to allow
time for toxin inactivation.
o After the allotted time, the decon solution from the labware soak must be retained, collected
and transferred to a secondary, leak-proof container and sealed.
 Fill out a yellow hazardous (chemical) waste label indicating concentration of
components in the waste bottle (including the amount and concentration of bleach) and
affix to the bottle.
 Store the doubly-contained inactivated toxin in the designated hazardous waste
accumulation area (fill in location). Do not store toxin waste for prolonged periods of
time. Toxin waste should be picked up ASAP as a best management practice.
 To schedule a hazardous waste pick up call EH&S (392-8400) or schedule online. EH&S Hazardous Waste will not pickup toxin materials that have not been
inactivated.
o Wash the re-usable glassware with soap and water prior to re-use.
11. Inactivation/Disposal of Liquid Stocks:
Describe any additional controls per your toxin risk assessment and/or the recommendation of the Biosafety
Office.
12. Spill Response:
Describe any additional controls and/or procedures per your toxin risk assessment and/or the recommendation of
the Biosafety Office.
13. Exposure Management:
Describe any additional controls and/or procedures per your toxin risk assessment and/or the recommendation of
the Biosafety Office.
14. Personnel Training:
Add any additional laboratory-specific training requirements to the statement already provided in the SOP
template.
For additional help in completing this SOP, contact the EH&S Biosafety Office at 392-1591.
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Appendix A: Sample Toxin Log Book Setup:
Toxin Name: _________________________________
Date Received: _______________________________
Source/Received From: _________________________
Form Received (Liquid, Powder): _________________
Total Amount Received (in mg): __________________
Concentration of Liquid Stock: ___________________
Total volume Liquid Stock: ______________________
Starting
Amount
(mg)
Amount
removed/used
(mg)
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Date of
Removal
Purpose
Removed by (Print name)
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Signature
Amount
remaining
(mg)
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Appendix B: Toxin in Use Sign
TOXIN IN USE
DO NOT ENTER
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Appendix C: Use of Biotoxins or Acute Toxins in Animals
Standard Operation Procedures for Handling Animals Dosed with (Fill in Toxin Name).
Note: This document is intended to be adapted to the lab as a site-specific document. Additional precautions or
regulations may be required per risk assessment by the Biological Safety Office and/or Institutional Biosafety
Committee and/or Responsible Official. Please add/subtract information as necessary to accurately reflect the
procedures specific to your project and avoid confusion.
A copy of this SOP should be provided to the Animal Care Services (ACS) and ACS staff should be provided with
information/training on the hazards associated with working with (fill in toxin name), required practices and
procedures and proper handling of bedding, cages and all other husbandry materials (e.g., carcass disposal)
associated with these experiments.
Use of all biotoxins in animals must be documented in a protocol approved by the UF Institutional Animal Care and
Use Committee (IACUC).
 IACUC approval #: fill in
Notification and Signage:
 When animals are dosed with (fill in toxin name), research personnel must clearly label the cages of the
animals with a “Chemical Hazard” sticker indicating the agent, date introduced and date cleared (the date
cleared will be determined by the project staff).
 For animals to require special care by research staff- place a “Special Care by PI” Card on the cage
indicating what the research staff will be responsible for (e.g., cage changing, feeding, water, etc).
o This card should include the name and phone number of the individual responsible for providing
o care.
o A Monthly Summary sheet must be completed by the researcher(s) documenting the husbandry
care provided by the research staff.
Personal Protective Equipment:
All personnel must wear appropriate personal protective equipment when handling animals, cages and bedding.
 For handling animals and cages (at a minimum): disposable long-sleeved lab coat, shoe covers, hair
covers, double gloves, surgical/dust masks, eye protection.
 For dumping bedding: the above listed PPE, plus an N95 respirator (with face shield, goggles, or safety
glasses) must also be worn.
Animal Innoculations:
 Preparation, Transport and Inoculation of Animals with toxin:
o All toxin doses must be prepared in a certified biological safety cabinet (BSC)
o If injecting the toxin:
 Load individual toxin doses into a single syringe; do not load multiple doses.
 Single-use, disposable luer-lok syringes should be used at a minimum.
 Safety syringe/needle combinations should be considered.
o If preparing toxin doses in your laboratory and transporting to the animal facility for dosing:
 Individual toxin doses will be loaded into syringes inside a BSC.
 Each toxin loaded syringe should be placed in a conical tube and secured using parafilm
for transport.
 Alternatively, a safety syringe/needle combination with a protective sheath
that has a transport position can be ordered and used.
 Enclosed syringes/needles will then be placed in a secondary hard-walled, sealable
container (e.g., Tupperware™) labeled with a Biohazard sticker, PI name and contact
number.
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
o
o
The outside surface of the secondary container will be decontaminated with freshly
prepared (insert decon solution, concentration) prior to removal from the BSC and
transport to the animal facility.
The buddy system will be used for all injections of toxin into animals.
For i.p. injections of toxins that are hazardous at low doses, anesthetizing animals should be
considered to minimize risk of self-inoculation.
 At a minimum, puncture resistant gloves will be worn underneath two pairs of
disposable gloves for all injections.
 After injections, the needle/syringe will be immediately placed in an appropriate
puncture-resistant sharps container.
 Sharps containers will be autoclaved immediately at the completion of the experiment.
Animal Cage-Change Procedures:
Engineering Controls:
 Animals dosed with (insert toxin name) must be maintained in microisolater cages with filter tops at
(insert ABSL level).
o Note: For animals too large to be housed in cages with microisolator lids or on ventilated racks,
you must consult with the Biological Safety Officer in advance to determine appropriate
engineering controls.
 Cages will be opened (including for cage-changing, animal care, or experiment-related reasons) in a
(choose the appropriate control: BSC, ventilated cage changing station, or chemical fume hood).
Work Practices:
 Contaminated, or potentially contaminated, bedding must be deactivated by the method listed in the
Waste Disposal section below.
 Outer gloves must be changed at least every two (2) hours, when they become torn or obviously
contaminated with excreta AND before handling animals in other experimental groups.
 All personnel will wash hands after removing gloves.
 Safety glasses, safety goggles, and reusable face shields will be decontaminated with freshly prepared
(insert decon solution, concentration) followed by a water rinse after use and stored in a clean location for
re-use.
 Biological safety cabinets will be surface-decontaminated after each use with freshly prepared (insert
decon solution, concentration) followed by a water rinse. Cleaning will proceed from least to most
contaminated area.
Waste Disposal Procedures:
Note: Research personnel must coordinate with the ACS Husbandry staff prior to the start of any experiment
involving inoculation of animals with a biotoxin or acute toxin to ensure that the appropriate procedures are
followed for cage changing and disposal of bedding and carcasses. Cages should be clearly marked to indicate any
special handling procedure.

For toxins that can be inactivated by autoclaving:
o Cages (with bedding and feed) will be double-bagged in red, biohazard-labeled, autoclavable
bags and taped shut.
o Bags will be sprayed with freshly prepared (insert decon solution, concentration) and allowed to
sit for a minimum of 5 minutes prior to removing from the BSC.
o Autoclave cages promptly when de-populated.
 The recommended cycle for inactivating heat-labile toxins is: 121°C, 15psi for 1 hour.
o Once autoclaved, bedding can be dumped according to standard practices and disposed of as
general trash.
o Animal carcasses will be placed in biohazard bags and incinerated.
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o

All disposable PPE will be discarded in a biohazard bag before leaving the work area and
subsequently autoclaved.
For toxins that cannot be inactivated by autoclaving:
o Collect contaminated bedding and feed in red biohazard-labeled bags.
 Label the bag “Animal bedding for incineration.”
o Place the bag into a biowaste box lined with another red biohazard-labeled bag and tape the box
shut at the completion of the experiment to be picked up by Stericyle for incineration.
o Label the box with a facility pre-printed biohazardous waste label.
o Submerge cages and water bottles in freshly prepared (insert decon solution, concentration) for a
minimum of 1 hr.
 Waste decon solution from the cage soak should be retained, collected and placed into
a secondary durable, leak-proof container and sealed.
 Fill out a yellow hazardous (chemical) waste label indicating concentration of
components in the waste bottle (including the amount and concentration of bleach) and
affix to the bottle.
 Store the doubly-contained inactivated toxin in the designated hazardous waste
accumulation area (fill in location). Do not store toxin waste for prolonged periods of
time. Toxin waste should be picked up ASAP as a best management practice.
 To schedule a hazardous waste pick up call EH&S (392-8400) or schedule online. EH&S Hazardous Waste will not pickup toxin materials that have not been
inactivated.
 Cages can then be washed according to standard procedures.
o Animal carcasses will be placed in biohazard bags and incinerated.
o Collect all disposable PPE in a biohazard bag before leaving the work area and package for
incineration.
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